Dulaglutide Protects Mice against Diabetic Sarcopenia-Mediated Muscle Injury by Inhibiting Inflammation and Regulating the Differentiation of Myoblasts

Background Type 2 diabetes mellitus increases the risk of sarcopenia, which is characterized by decreased muscle mass, strength, and function. However, there are no effective drugs to treat diabetic sarcopenia, and its underlying mechanism remains unknown. Here, we aimed to determine whether the GLP-1 receptor agonist (GLP-1RA) dulaglutide (Dul) affects the progression of diabetic sarcopenia. Methods db/db mice were injected intraperitoneally with 0.6 mg/kg dulaglutide for 10 weeks. Mouse muscle tissues were then pathologically evaluated and stained with F4/80 or MPO to detect macrophages and neutrophils, respectively. In addition, inflammatory factors and FNDC5 in the muscle tissues were detected using qRT-PCR. Moreover, C2C12 cells were induced to enable their differentiation into skeletal muscle cells, and muscle factor levels were then detected. Furthermore, changes in muscle factor levels were detected at various glucose concentrations (11 mM, 22 mM, and 44 mM). Results In vivo, dulaglutide alleviated muscle tissue injury; reduced levels of the inflammatory factors, IL-1β, IL-6, CCL2, and CXCL1; and reversed the level of FNDC5 in the muscle tissues of db/db mice. In vitro, a C2C12 cell differentiation model was established through the observation of cell morphology and determination of myokine levels. Upon stimulation with high glucose, the differentiation of C2C12 cells was inhibited. Dulaglutide improved this inhibitory state by upregulating the levels of both FNDC5 mRNA and protein. Conclusions Treatment with the GLP-1RA dulaglutide protects db/db mice against skeletal muscle injury by inhibiting inflammation and regulating the differentiation of myoblasts. High glucose inhibited the differentiation of C2C12 cells and decreased the mRNA and protein levels of myokines. Dulaglutide could reverse the differentiation state induced in C2C12 cells by high glucose.


Introduction
Te prevalence of diabetes mellitus is gradually increasing. Te numbers of individuals sufering from diabetes in China and India were 145 million and 74 million, respectively, in 2021, accounting for 41% of the global adult diabetic population [1]. Sarcopenia is a muscle-wasting syndrome characterized by the progressive and systemic degenerative loss of skeletal muscle mass and strength [2,3]. Both aging and Type 2 diabetes mellitus (T2DM) are risk factors for sarcopenia, and the risk of sarcopenia is signifcantly increased in elderly patients with T2DM. Studies have shown that the prevalence of sarcopenia in elderly patients with T2DM is 2-3 times higher than that in the average population and can reduce physical activity and daily activities, seriously afecting the quality of life and increase the hospitalization rate, medical expenses, and risk of death in the elderly [4][5][6]. Terefore, new therapeutic strategies that can improve T2DM with sarcopenia are awaited.
Diabetes and sarcopenia afect each other. Glucose metabolism disorders promote catabolism and muscle protein decomposition, leading to decreased muscle function and muscle content [7]. Te decrease in muscle content further aggravates insulin resistance in muscles, which inhibits energy metabolism in the mitochondria of muscle cells and afects the normal contractile function of muscle tissue [8]. Both myokines and infammation play an essential role in the pathogenesis of T2DM and sarcopenia [9]. Various infammatory cells release pro-infammatory mediators in skeletal muscles. Subsequently, muscle cells are damaged or even die, resulting in the loss of muscle contractile properties [10]. Te myokines secreted by skeletal muscles include myostatin, Fibronectin Type III Domain-containing Protein 5 (FNDC5), insulin-like growth factor 1 (IGF1), fbroblast growth factor 21 (FGF21), etc. [11]. At present, besides conventional lifestyle interventions, namely physical exercise and caloric control, calcium and vitamin D supplements, mesenchymal stem cell therapy, and myostatin inhibitors, are used for the treatment of diabetic sarcopenia [12][13][14]. However, none of these can improve sarcopenia to a great extent. Terefore, it is urgent to fnd new means to treat sarcopenia.
Glucagon-like peptide-1 receptor agonists (GLP-1RAs) have been developed as an anti-diabetic therapy to promote insulin secretion in T2DM [15]. A study reported that the activation of GLP-1R signaling may be helpful in the treatment of atrophy-related muscle diseases [16]. A recent study found that GLP-1RAs can recover muscle weakness by suppressing muscle infammation and muscle fber necroptosis [17]. We therefore hypothesized that GLP-1RAs exert benefcial efects on T2DM with sarcopenia to recover muscle strength, suppress muscle infammation, and regulate myokines. Dulaglutide, as a weekly preparation of GLP-1, has been shown to be benefcial in improving diabetes mellitus associated with chronic kidney disease as well as cardiovascular disease in clinical trials [18,19]. Here, we show that dulaglutide protects against T2DM with sarcopenia by inhibiting infammation and regulating the diferentiation of myoblasts using in vivo and in vitro models of T2DM with sarcopenia.

Materials and Methods
2.1. Cell Culture and Treatment. C2C12 cells were cultured in Dulbecco's modifed Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) at 37°C in a humidifed atmosphere containing 5% CO 2 . In the exponential growth phase, the cells were inoculated into a 6-well plate (4 × 10 5 /well) and incubated at 37°C for 24 h. Te culture medium was discarded at this point, and 2 mL of diferentiation medium (containing 2% horse serum, 97% DMEM, and 1% penicillin-streptomycin) was added. After 4 days of culture, morphological changes in the C2C12 cells were observed using microscopy. Similarly, diferent concentrations of glucose (11 mM, 22 mM, and 44 mM) and 100 nM dulaglutide were added to diferentiated cells, according to the assigned group treatment, and cells were collected 24 h later for further analysis.

Sulforhodamine B (SRB) Colorimetric
Assay. C2C12 cells in the logarithmic phase were plated in 96-well plates containing 8 × 10 3 cells per well. At the end of the drug action time, 50% TCA solution was added to each well to fx the cells, and the cells were then transferred to a refrigerator at 4°C for overnight incubation. After drying, 70 μL of 0.4% SRB staining solution was added to each well. After staining for 30 min, the staining solution was poured out, and the cells were rinsed with 1% acetic acid fve times and then dried at room temperature. Te dye was dissolved in 100 μL of non-bufered tris-base lye (10 mM, pH � 10.5), and the light absorption value in each well was measured at 540 nm using a microplate reader.

Animal Treatment.
Specifc pathogen-free (SPF) male diabetic db/db mice and nondiabetic littermate db/m mice (10 weeks) were purchased from Nanjing Mairuisi Biotechnology. All mice were kept in a controlled environment, allowed ad libitum access to food and water, and habituated to the room used to house them for 7 days before being subjected to experiments. After adaptation, the mice were divided randomly into four groups: a control group (db/m, 0.9% saline), a model group (db/db, 0.9% saline), a treatment group (db/db, dulaglutide 0.6 mg/kg), and a drug control group (db/m, dulaglutide 0.6 mg/kg), with six animals in each group. Te Dul dosage selection was determined based on previous relevant experiments [20]. Drugs were administrated using intraperitoneal injection each week. For the experiment, one drop of mouse blood was collected using tail vein puncture, and the blood glucose level was measured weekly using a blood glucose meter. All the mice were euthanized 10 weeks after drug treatment. Blood samples were collected at the time of euthanasia. Muscle tissue was partly fxed in 4% phosphate-bufered formaldehyde for histological analyses and partly snapped frozen for subsequent molecular analysis. All animal experiments were performed following the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Center for scientifc research at the Second Afliated Hospital of Anhui Medical University (20211123).

Histologic Evaluation.
Muscle sections (5 μm) were stained with hematoxylin & eosin (H&E). Briefy, fresh muscle tissues were collected, fxed with 4% paraformaldehyde, embedded in parafn, and sliced. Te tissue slices were then stained with HE stain to observe histological changes in the muscles. Muscle injury was evaluated by a pathologist in a blinded manner and was assessed using the Manickam R method, as previously described [21]. Images were obtained using an Olympus BX41 microscope.

Immunohistochemistry.
After deparafnization and rehydration, 0.01 M citrate bufer (pH 6.0) was added to the tissue sections for antigen retrieval. Ten, the sections were blocked with 10% goat serum for 30 min at room temperature. Next, the sections were incubated with F4/80 (Afnity, cat number: DF2789, 1 : 300) and MPO (Proteintech, cat number: 22225-1-AP, 1 : 400) antibodies overnight at 4°C. Subsequently, an HRP-DAB system (ZSGBBIO, Beijing, China) was used to detect immunoactivity in the tissue sections. Tis was followed by counterstaining with hematoxylin. After stepwise dehydration, the tissue sections were sealed, and their images were obtained using an Olympus BX41 microscope.

Quantitative Real-Time PCR.
Total RNA was extracted from the treated muscle tissues using the TRIzol reagent (Termo Fisher Scientifc, Waltham, USA) and transformed to cDNA using the HyperScriptTM III 1st Stand cDNA Synthesis Kit (NovaBio, Shanghai, China). Quantitative realtime PCR was performed using the S6 Universal SYBR qPCR mix (NovaBio, Shanghai, China) and an ABI 7900 PCR system (ABI, USA). Te 2 −ΔΔCt method was used to assess relative gene expression, and GAPDH was used for normalization of data. All primers were custom-made by Genscript. Primer sequences are listed in Supplementary  Table 1.

Statistical
Analysis. Data are expressed as mean ± SEM, and statistical analyses were performed using GraphPad Prism 8.0. Statistical signifcance of diferences between two groups was analyzed using Student's t-test and that of differenced between more than two groups was analyzed using one-way ANOVA followed by Turkey's multiple comparison test. P < 0.05 was considered statistically signifcant.

Dulaglutide Inhibited Diabetes-Induced Muscle Atrophy.
After being administered dulaglutide for 10 weeks, the db/db mice were dissected, and their soleus muscles were fxed, sectioned, and subjected to HE staining. Statistical analysis showed that the average cross-sectional area of db/db mouse soleus muscles was signifcantly decreased compared to that of age-matched db/m mouse soleus muscles, but this reduction in the cross-sectional area of db/db mouse soleus muscles could be inhibited by dulaglutide (Figures 1(a) and  1(b)). Contrarily, the mRNA level of FNDC5 in db/db mouse soleus muscles of was signifcantly decreased compared to that in db/m mouse soleus muscles; however, treatment with dulaglutide increased the expression of FNDC5 in the soleus muscles of both db/m and db/db mice (Figure 1(c)).

Dulaglutide Inhibited the Infammatory Response in
Diabetes-Induced Muscle Atrophy. Infammation is often involved in the pathogenesis of sarcopenia. Terefore, we used qRT-PCR to measure the expression of a series of infammatory factors and chemokines. Te mRNA levels of IL-1β and IL-6 in skeletal muscles were not notably diferent between db/m and db/db mice but were decreased upon dulaglutide administration in db/db mice (Figure 2(a)). In addition, the mRNA levels of CCL2, CXCL1, and CXCL2 in skeletal muscles were markedly increased in db/db mice compared to those in db/m mice. Te mRNA levels of CCL2 and CXCL1 were signifcantly reduced in db/db mice upon treatment with dulaglutide, although the mRNA level of CXCL2 did not change signifcantly upon treatment with dulaglutide ( Figure 2(b)).

Dulaglutide Attenuated Infammatory Cell Infltration in
Diabetes-Induced Muscle Atrophy. In addition to changing the levels of infammatory factors in the blood, chemokines also induce infammatory cells to migrate and infltrate into tissues and organs secreting them. Neutrophils are the earliest infltrating infammatory cells in acute infammation, while macrophages are the primary functional cells involved in chronic infammation. Our immunohistochemistry results showed that, compared with those in db/ m mouse soleus muscles, the levels of the neutrophil marker MPO and macrophage marker F4/80 in db/db mouse soleus muscles were signifcantly increased; these levels were signifcantly decreased in db/db mice after they were treated with dulaglutide (Figures 3(a) and 3(b)).

Efect of Diferent Glucose
Concentrations on the Proliferation and Diferentiation of C2C12 Cells. C2C12 cells were stimulated with 2% horse serum to diferentiate them into myotubes and increase the mRNA levels of the myocyte diferentiation genes MyHC4 and MyHC7 and myokine factors MyoD and MyoG in them (Figures S1(a)-S1(c)). To analyze the efect of glucose on myocyte diferentiation, diferentiated mouse C2C12 myotubes were stimulated with diferent concentrations of glucose (11 mM, 22 mM, and 44 mM). Te SRB assay was used to determine whether glucose had any efect on the viability of C2C12 cells. Te results showed that high glucose promoted the proliferation of C2C12 cells, and cell proliferation was weakened with a further increase in the glucose concentration. Te efect of 11 mM glucose on C2C12 cell proliferation was the most signifcant (Figure 4(a)). However, after stimulati n with a high glucose concentration, the mRNA levels of the diferentiation-related gene MyHC4, MyHC7, the myogenic factor MyoD, and the muscle secretory factors FNDC5 and BDNF were decreased in diferentiated C2C12 cells (Figures 4(b)-4(d)).  (c) RNA was extracted from the soleus muscles, and the mRNA levels of FNDC5 were detected. * * * P < 0.001, * comparison between the db/db group and control db/m group; # P < 0.05, ## P < 0.01, # comparison between the dulaglutide-treated group and db/db group.

Expression of Myostatin, PGC-1α, and FNDC5 in C2C12 Cells Treated with Diferent Glucose Concentrations.
C2C12 cells were diferentiated and stimulated with glucose at diferent concentrations (11 mM, 22 mM, and 44 mM). Proteins were then extracted from C2C12 cells stimulated with varying concentrations of glucose (11 mM, 22 mM, and 44 mM). Te expression level of the myostatin protein increased in response to high-glucose stimulation, while the expression levels of the FNDC5 and PGC1-α proteins decreased (Figures 5(a) and 5(b)). Tese results indicated that high glucose concentrations afected the expression levels of muscle factors in C2C12 cells.

Efect of Dulaglutide on C2C12 Cells Treated with High
Concentrations of Glucose. To investigate the efect of dulaglutide on C2C12 cells stimulated by high glucose, we administered 100 nM dulaglutide and 11 mM glucose to diferentiated C2C12 cells simultaneously and measured mRNA and protein expression levels after 24 h of culture.
Te high glucose concentration increased the mRNA levels of MyHC4, MyHC7, and FNDC5 and the protein level of FNDC5 in diferentiated C2C12 cells, while dulaglutide decreased these levels (Figures 6(a) and 6(b)). Tese fndings indicated that dulaglutide could reverse the efects of high glucose concentrations on myocyte diferentiation and the FNDC5 expression, suggesting that high glucose concentrations could afect the diferentiation of C2C12 cells, and dulaglutide could improve myocyte diferentiation and increase the efect of FNDC5.

Discussion
In the present study, we frst found that dulaglutide improved pathological changes in muscles and inhibited infammation related to diabetic sarcopenia in db/db mice. Moreover, we found that dulaglutide reversed the reduction in the FNDC5 mRNA level induced by high glucose concentrations in db/db mice. Next, we investigated whether the diferentiation of C2C12 cells into skeletal muscle cells could  Figure 2: Dulaglutide inhibited the infammatory response in mice with diabetes-induced muscle atrophy. (a-b) Te mRNA levels of IL-1β, IL-6, TNF-α, CCL2, CXCL1, and CXCL2 in soleus muscles were measured using qRT-PCR. * P < 0.05, * comparison between the db/db group and control db/m group; # P < 0.05, ### P < 0.001, # comparison between the dulaglutide-treated group and db/db group.
International Journal of Endocrinology 5 be induced by high glucose concentrations. We found that dulaglutide could improve myocyte diferentiation in C2C12 cells stimulated with high concentrations of glucose.
Tese results suggest that dulaglutide may protect against diabetic sarcopenia by regulating infammation and myokine levels. Type 2 diabetes mellitus is associated with accelerated muscle loss during aging, decreased muscle function, and increased disability [22]. As a weekly preparation of GLP-1RA, dulaglutide has been benefcial in improving the condition of diabetic patients with chronic kidney disease and cardiovascular disease in clinical trials [18,19]. Some studies have found that GLP-1RA may afect skeletal muscles by afecting the expression of myokines. Te GLP-1RA PF1801 can improve muscle weakness in polymyositis and suppress muscle infammation by inhibiting muscle fber necroptosis [17]. Animal studies have shown that dulaglutide improves muscle mass and strength, inhibits muscle atrophy factor, and increases the expression of the myogenic factor MyoD in aged mice, suggesting that dulaglutide may play a benefcial role in the treatment of muscle atrophy [23]. Fibronectin III domain containing 5 (FNDC5) is a type I transmembrane glycoprotein, the proteolysis of which at the carboxyl terminus releases irisin, which mainly comprises the fbronectin III domain of FNDC5 [24]. A meta-analysis found a direct positive correlation between serumcirculating irisin and insulin resistance in non-diabetic adults [25]. Trough the assessment of body weight, body fat, and endothelial cell dysfunction markers, one study found that the increased plasma irisin level in patients with type 2 diabetes was correlated with the levels of obesity indicators, and irisin may be involved in atherosclerotic endothelial injury accompanied by obesity and type 2 diabetes mellitus [26].
Recent studies have shown that the myokine irisin afects bone metabolism in vivo. Mice treated with irisin showed improvements in cortical bone mass, geometry, and strength, similar to how physical activity afects the development of adequate weight-bearing bone. Irisin is a potential biomarker of muscle dysfunction that can help predict the onset of sarcopenia and provide a new way to monitor age-related muscle changes [27].
One study investigated the efect of the GLP-1RA exenatide on irisin levels in newly diagnosed obese patients with T2DM. Changes in the irisin level after treatment with exenatide were associated with decreases in FBG and HbA1c levels. Upregulation of irisin may be a new mechanism underlying the efect of exenatide in the treatment of type 2 diabetes mellitus [28]. Nother study explored changes in serum irisin and IL-6 levels in patients with T2DM after 6 and 12 months of treatment with a GLP-1RA. Further treatment with GLP-1 analogues increased the serum-circulating irisin level and decreased the IL-6 level. Changes in the irisin level after treatment with GLP-1 were associated with a decrease in total cholesterol, while changes in the IL-6 level were associated with a reduction in the waist circumference [29]. In the present study, the level of serum-circulating irisin in patients with diabetes mellitus was reduced, consistent with previous studies.  Figure 3: Dulaglutide attenuated infammatory cell infltration in diabetes-induced muscle atrophy. (a, b) Te macrophage marker F4/80 and neutrophil marker MPO in the soleus muscles of mice were stained and their expression levels were analyzed using immunohistochemistry. Te scale bars represent 100 μm and 50 μm, respectively. * * * P < 0.001, * comparison between the db/db group and control db/m group; ## P < 0.05, # comparison between the dulaglutide-treated group and db/db group.
It has been reported that a high Neutrophil/Lymphocyte Ratio (NLR) is associated with the risk of sarcopenia in hospitalized patients with cancer [30,31]. A multicenter prospective longitudinal sarcopenia study was conducted in the Peking Union Medical College Hospital and involved 56 elderly patients with sarcopenia and 56 elderly nonsarcopenia patients. Te study found that, compared with those in non-sarcopenia patients, the serum levels of IL-6, IL-18, TNF-α, TNF-like weak inducing factor of apoptosis (TWEAK), and leptin were signifcantly increased in sarcopenia patients [32]. In the skeletal muscle of TNFα-overexpressing transgenic mice, TNF-α levels were increased, accompanied by muscle atrophy, reduced numbers of satellite cells and type IIa muscle fbers, and an increased number of myeloid cells, including macrophages and granulocytes. An increased expression of TNF-α in myeloid cells impairs their diferentiation [31].
Overall, in the present study, we examined the expression levels of the infammatory cytokines IL-1β, IL-6, and TNF-α and the infammatory chemokines CCL2, CXCL1, and CXCL2 in the muscle tissues of aged db/db mice before and after dulaglutide intervention. Te results showed that the levels of CCL2, CXCL1, CXCL2, IL-1β, and IL-6 in the muscle tissues of db/db mice were signifcantly increased. Te increase in these levels was not signifcant, which may be related to individual diferences. Dulaglutide could signifcantly reduce these increased levels of IL-1β, IL-6, CCL2, and CXCL1. Increased levels of chemokines can recruit a large number of infammatory cells, such as macrophages and neutrophils, to aggravate muscle tissue damage, and dulaglutide can reverse this infammatory cell infltration efect, suggesting that dulaglutide can reduce muscle tissue injury in aged diabetic mice, partly by inhibiting infammation. Tis is similar to our previous study [33].
Te present study has the following limitations. First, the treatment efects of dulaglutide on diabetic sarcopenia patients is unknown and need further investigation. Second, due to the irisin release caused by FNDC5, the level of irisin under dulaglutide treatment should be determined in order to research the relationship between myokines and diabetic sarcopenia in depth. In summary, our present study showed the protection efect of dulaglutide against muscle tissue injury in mice with diabetic sarcopenia by inhibiting infammation and regulating the diferentiation of myoblasts. In addition, we hope that our study can provide new

Data Availability
All the data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Conflicts of Interest
Te authors declare that they have no conficts of interest.